Rho GTPases activated by upstream signals coordinate the cytoskeletal dynamics necessary for such complex motile processes as chemotaxis, metastasis, axon guidance, etc. One critical mechanism by which Rho GTPases control actin dynamics is via regulation of the action of cofilin/ADF family proteins. Cofilin/ADF (C/A) proteins act to sever F-actin filaments and promote F-actin depolymerization, both activities that are critical to the cytoskeletal rearrangements involved in formation of leading edge lamellae and cell motility. The action of C/A is regulated by phosphorylation/dephosphorylation of a critical regulatory site at Ser3. We have previously shown that Rac and Cdc42 regulate the phosphorylation and inactivation of C/A through the action of a downstream kinase cascade involving p21-activated kinase (PAK) and LIM kinase. We have now identified a unique phosphatase that acts to dephosphorylate and thus activate C/A. In combination with biochemical studies, we will utilize genetic means and RNA interference methodologies to disrupt phosphatase function in vivo. We propose to investigate the properties and regulation of this novel CA phosphatase in order to determine its importance in modulating cytoskeletal dynamic behavior. We have discovered regulatory interactions between Ser3-phosphoCA and 14-3-3 family proteins. We will investigate the hypothesis that, in addition to direct regulatory effects on CA, 14-3-3 proteins act as intermolecular scaffolds to link CA regulatory kinases and phosphatases into a coordinated regulatory module. Finally, we will investigate the coordinated action of the PAK1-LIMK-CA phosphatase-CA pathway during stimulated cell motility. Regulatory events modulating this signaling pathway will be determined. The question of localized vs. global regulation of CA function will be addressed by real time studies of the spatial and temporal dynamics of the signaling molecules involved. The proposed studies should give us unique information about the action of Rho GTPases to regulate CA function during cell motility.